Isotachophoresis
Isotachophoresis (“ITP”) is a variant of electrophoresis, characterized by the fact that separation is carried out in a discontinuous buffer system. Sample material to be separated is inserted between a “leading electrolyte” and a “terminating electrolyte” or mixed in any of these, the characteristic of these two buffers being that the leader has to have ions of net mobility higher than those of sample ions, while the terminator must have ions of net mobilities lower than those of sample ions. In such a system, sample components sort themselves according to decreasing mobilities from leader to terminator, in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described repeatedly, as for instance, Bier and Allgyer, Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979).
It is further characteristic of ITP that a steady state is eventually reached, where all components migrate at same velocity (hence the name) in sharply defined contiguous zones. Sample components can be separated in such a contiguous train of components by insertion of “spacers” with mobilities intermediary between those of the components one wishes to separate.
Isoelectric focusing (“IEF”), also sometimes called electrofocusing, is a powerful variant of electrophoresis. The principle of IEF is based on the fact that proteins and peptides, as well as most biomaterials, are amphoteric in nature, i.e., are positively charge in acid media and negatively charged in basic media. At a particular pH value, called the isoelectric point (PI), there is reversal of net charge polarity, the biomaterials acquiring zero net charge.
If such amphoteric materials are exposed to a d.c. current of proper polarity in a medium exhibiting a pH gradient, they will migrate, i.e., ‘focus’ toward the pH region of their PI, where they become virtually immobilized. Thus a stationary steady state is generated, where all components of the mixture have focused to their respective PIs.
The pH gradient is mostly generated ‘naturally’ i.e, through the electric current itself. Appropriate buffer systems have been developed for this purpose, containing amphoteric components which themselves focus to their respective PI values, thereby buffering the pH of the medium.
The two variants, IEF and ITP, differ in that IEF attains a stationary steady state whereas in ITP a migrating steady state is obtained. Thus, in IEF a finite length of migrating channel is always sufficient. In ITP, complete resolution may require longer migrating channels than is practical. In such case, the migrating components can be virtually immobilized by applying a counterflow, the rate of counterflow being matched to the rate of frontal migration of the sample ions. This is also known in the art.
IEF is most frequently carried out in polyacrylamide or agarose gels, where all fluid flow disturbances are minimized. ITP is most often carried out in capillaries. The sample is inserted at one end of the capillary, at the interface between leader and terminator, and the migration of separated components recorded by appropriate sensors at the other end of the capillary. Both such systems are used mainly for analytical or micro-preparative purposes.
ITP forms a sharp moving boundary between ions of like charge. The technique can be performed with anionic or cationic samples. The system quickly establishes a strong gradient in electric field at the ITP interface, due to the non-uniform conductivity profile. As per its name (from Greek, “isos” means “equal”, “takhos” means “speed”), TE and LE ions travel at the same, uniform velocity, as a result of the non-uniform electric field and conservation of current (this is the so-called “ITP condition”).
The ITP interface is self-sharpening: LE ions that diffuse into the TE zone experience a strong restoring flux and return to the leading zone (and vice versa for TE ions in the LE zone). Sample ions focus at this interface if their effective mobility in the TE zone is greater than those of the TE co-ions, and if their effective mobility in the LE zone is less than that of the LE co-ions. The self-sharpening and focusing properties of ITP contribute to the robustness of this technique and make ITP relatively insensitive to disturbances of the interface (e.g. due to pressure-driven flow or changes in geometry, such as contractions, expansions, and turns).
In peak mode ITP, sample ion concentrations are at all times significantly lower than LE and TE ion concentrations and therefore contribute negligibly to local conductivity. The distribution of sample ions is determined by the self-sharpening interface between neighboring zones (here the TE and LE) and the value of the sample effective mobility relative to these zones. Multiple sample ions focus within the same narrow ITP interface region as largely overlapping peaks.
Pathogen Detection
The conventional bacteria detection methods—e.g. sample cultivation, genotypic detection methods and immunoassays—are time consuming, comprise of several manual steps, and require highly trained personnel. In recent years, there has been significant interest in the use of microfluidic platforms for pathogen detection. Microfluidic technology enables the manipulation and analysis of small volumes of sample, typically on the order of several nl to several μl and can be leveraged toward rapid and highly sensitive analysis.
Oukacine et al. (Anal. Chem. 2011, 83, 4949-4954) used simultaneous electokinetic and hydrodynamic injection with UV detection and thus required no labeling. Prior to injection, sample was filtered and isolated from the original water matrix and then resuspended in a low conductivity electrolyte which was then used in the analysis. This method provided a limit of detection of 2×104 cfu/mL.
Another approach was explored by Phung et al. (Electrophoresis 2013, 34, 1657-1662) in order to improve the sensitivity of detection. Their assay involves a prelabeling step in which the sample was incubated with SYTO9 dye (a cell permeable nucleic acid stain) for approximately 30 min. This was followed by ITP focusing of bacteria from the sample and fluorescence detection of the formed peak. The assay was performed in a standard capillary electrophoresis apparatus and achieved a limit of detection of 135 cfu/mL. The authors have also demonstrated the detection of bacteria at a concentration of ˜104 cfu/mL from contaminated river water samples, after filtering the sample to remove particulates.
However, despite the many advantages of this technology, to date, most microfluidic assays cannot perform continuous analysis and are limited by their ability to analyze only a single and finite amount of sample, and are thus coupled to significant sample preparation, e.g. by filtration or centrifugation. This is in contrast to the need for continuous and real time monitoring in many pathogen detection applications. Thus, there is an unmet need for rapid, continuous, effective, portable and more accurate detection and identification of infectious disease-causing pathogens, with the potential of automation and standardization.